WO2020224364A1 - Mcl1蛋白抑制剂在制备治疗炎症及神经退行性疾病药物中的应用 - Google Patents

Mcl1蛋白抑制剂在制备治疗炎症及神经退行性疾病药物中的应用 Download PDF

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WO2020224364A1
WO2020224364A1 PCT/CN2020/083442 CN2020083442W WO2020224364A1 WO 2020224364 A1 WO2020224364 A1 WO 2020224364A1 CN 2020083442 W CN2020083442 W CN 2020083442W WO 2020224364 A1 WO2020224364 A1 WO 2020224364A1
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inflammation
disease
mcl1
umi
protein inhibitor
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夏宏光
岑旭峰
许正平
盛静浩
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Zhejiang University
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Zhejiang University
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    • C12N2740/10011—Retroviridae
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Definitions

  • the invention belongs to the technical field of inflammation treatment, and specifically relates to the application of UMI-77 and its derivatives as mitochondrial autophagy inducers in the preparation of drugs for treating inflammation and neurodegenerative diseases.
  • Mitophagy plays an important role in the inhibition of inflammation. Inflammation is the basic strategy of the body to fight infection, and it is also the main cause of exacerbation of diseases, such as certain neurodegenerative diseases and Alzheimer's disease. Therefore, screening new safe and effective mitochondrial autophagy inducers is a possible therapeutic strategy to inhibit inflammation and alleviate the disease.
  • the main principle of the existing mitochondrial autophagy inducers is to destroy the function of mitochondria and force cells to undergo mitochondrial autophagy. Although it can induce mitochondrial autophagy, it cannot play a role in clinical applications due to its greater toxicity.
  • MCL1 inhibitors can be used as mitochondrial autophagy inducers, and there are no drugs targeting mitochondrial autophagy inducers or MCL1 protein inhibitors to treat inflammation and Alzheimer's disease.
  • the purpose of the present invention is to provide a mitochondrial autophagy inducer and its application.
  • an MCL1 protein inhibitor for the preparation of a medicine or composition for one or more purposes selected from the following group:
  • the inflammation includes inflammation in a tissue selected from the group consisting of adipose tissue, kidney tissue, liver tissue, muscle tissue, skin tissue, pancreas tissue, lung tissue, blood vessel, heart, bone, brain, spinal cord , Retina, gastrointestinal tissue, reproductive system, or a combination thereof.
  • the inflammation includes hepatitis (such as acute hepatitis), enteritis (such as ulcerative enteritis), autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis), metabolic disease inflammation (such as diabetes caused Nephritis).
  • hepatitis such as acute hepatitis
  • enteritis such as ulcerative enteritis
  • autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis
  • metabolic disease inflammation such as diabetes caused Nephritis.
  • the neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), different types of spinocerebellar ataxia (SCA), Pick disease.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • HD Huntington's disease
  • ALS amyotrophic lateral sclerosis
  • MS multiple sclerosis
  • SCA spinocerebellar ataxia
  • the MCL1 protein inhibitor includes a compound having MCL1 protein inhibitory activity disclosed in patent document US2014/0235702A1.
  • the MCL1 protein inhibitor is a compound with the structure shown in Formula I or a pharmaceutically acceptable salt thereof:
  • R1 is X is halogen
  • R2 is R 3 is H, halogen, or hydroxyl.
  • the MCL1 protein inhibitor is compound UMI-77 or UMI-59:
  • the medicine or composition contains a safe and effective amount of MCL1 protein inhibitor and a pharmaceutically acceptable carrier.
  • the drug or composition further includes an antioxidant.
  • the drug or composition is in unit dosage form.
  • the drug or composition includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.
  • the medicine or composition includes tablets, pills, powders, granules, capsules, syrups, emulsions, suspensions, injections, patches, drops, ointments, suppositories or sprays.
  • the administration method of the drug or composition includes oral, intramuscular injection, intravenous injection, intravenous drip, spray, or intraperitoneal injection.
  • the safe and effective amount refers to 5-1000 mg/kg; preferably 50-1000 mg/kg.
  • the safe and effective amount includes 50-200 mg/kg (mouse), or 500-1000 mg/kg (human).
  • the safe and effective amount is 100-700 mg/kg, more preferably 500-700 mg/kg.
  • the second aspect of the present invention provides a method for inducing mitochondrial autophagy, which includes the steps of: administering an MCL1 protein inhibitor to a subject in need (such as cells cultured in vitro).
  • the MCL1 protein inhibitor is as described above.
  • the method is an in vitro non-therapeutic method.
  • the concentration of the MCL1 protein inhibitor is about 0.001 ⁇ M-1000 ⁇ M, preferably about 0.1 uM-100 ⁇ M.
  • the third aspect of the present invention provides a method for promoting the interaction between MCL1 protein and LC3A protein, which includes the step of applying an MCL1 protein inhibitor in a system comprising MCL1 protein and LC3A protein.
  • the MCL1 protein inhibitor is as described above.
  • the system containing MCL1 protein and LC3A protein is a cell culture medium.
  • the fourth aspect of the present invention provides a composition for inhibiting inflammation or treating inflammation-related diseases, the composition comprising an MCL1 protein inhibitor.
  • the MCL1 protein inhibitor is as described above.
  • the composition further includes a pharmaceutically acceptable carrier.
  • the composition further includes an antioxidant.
  • the fifth aspect of the present invention provides a method for preventing or treating neurodegenerative diseases, the method comprising the steps of: administering a safe and effective amount of an MCL1 protein inhibitor to a subject in need.
  • the MCL1 protein inhibitor is as described above.
  • Figure 1 shows whether the HEK293Tmtkeima system of human embryonic kidney transformed cells can indicate mitochondrial autophagy according to the present invention
  • Figure 2 shows the high-throughput screening of more than 2000 FDA-approved drugs in the present invention
  • the intensity of the color represents the contrast between the 586nm excitation fluorescence and the negative control
  • the darker the color represents the stronger the induction of mitochondrial autophagy
  • Figure 3 is the second verification of 33 positive drugs of the present invention.
  • the bar graph represents the degree of mitochondrial autophagy induction, and the higher the induction, the stronger the induction;
  • the solid line is the negative control baseline, and the dashed line is the 1.5-fold increase line;
  • FIG. 4 shows the UMI-77 of the present invention induces degradation of mitochondrial marker proteins Tom20 and Tim23;
  • Fig. 5 is the result of Caspase 3 detected by UMI-77 processing cell immune hybridization (western blot) of the present invention
  • Figure 6 is the result of mitochondrial autophagy induced by the HEK293Tmtkeima system of human embryonic kidney transformed cells of the present invention under UMI-77 treatment; the image is taken by biotek cytation3, magnification 4X; red (the upper and lower images corresponding to EM586) is mtkeima586nm excitation fluorescence, representative Mitochondria where mitochondrial autophagy occurs. Green (the two upper and lower pictures corresponding to EM469) is 488nm excitation fluorescence representing normal mitochondria;
  • Fig. 7 is a laser confocal picture of live cells after HEK293Tmtkeima cells are treated with UMI-77 to observe the co-localization of mitochondria and lysosomes;
  • Figure 8 shows the degradation of mitochondria through lysosomes caused by treatment of HEK293T and Hela cells with UMI-77 of the present invention
  • Fig. 9 is the result of mitochondria after UMI-77 treatment of HEK293T cells by transmission electron microscope of the present invention.
  • Figure 10 is the results of the determination of the activity of alanine aminotransferase and aspartate aminotransferase in the blood of the mice of the present invention.
  • UMI-77 inhibits hepatocyte damage;
  • Figure 11 shows the HE (hematoxylin-eosin) stained mouse liver section of the present invention showing that UMI-77 restores the normal morphology of liver cells;
  • Figure 12 shows the MCL1 protein required for UMI-77 to induce mitochondrial degradation of the present invention
  • Figure 13 is the present invention In Situ Red Starter Kit Mouse/Rabbit kit (purchased from Sigma-Aldrich, #DUO92101) to detect the interaction between MCL1 and LC3A;
  • Figure 14 is the immunoprecipitation experiment of the present invention showing that UMI-77 induces enhanced interaction between LC3A and MCL1;
  • Figure 15 is the immunoprecipitation of the present invention showing that the interaction between MCL1 and LC3A is achieved through LIR motif;
  • Figure 16 is a mouse inflammatory bowel disease model of the present invention and a flow chart of UMI77 treatment experiment;
  • Figure 17 is the weight statistics results of mice of the present invention.
  • UMI77 can significantly slow down the weight loss, suggesting that it can protect inflammatory bowel disease;
  • Figure 18 is the statistical result of the disease activity index of the mouse of the present invention, which is calculated based on the weight change, blood in the mouse stool and stool characteristics;
  • Fig. 19 is a representative diagram of the colorectal in the mouse of the present invention, and the statistical results of the length of the colorectal in the mouse;
  • Figure 20 is a HE staining image of the colorectal of a mouse of the present invention, and histopathological score of the colorectal of the mouse;
  • Figure 21 is the expression level of inflammatory factors in the colorectal tissue of the mouse of the present invention.
  • Figure 22 is a record of the escape latency of each group of mice in the positioning cruise experiment of the present invention.
  • Figure 23 is the space search experiment of the present invention to record the number of times the mouse has passed through the original platform
  • Figure 24 is the movement track of a representative mouse recorded in the space search experiment of the present invention.
  • MCL1 protein inhibitors can induce mitochondrial autophagy and promote the interaction between MCL1 protein and LC3A protein.
  • Experimental results show that MCL1 protein inhibitors have a significant inhibitory effect on inflammation, and can be used for the prevention or treatment of inflammation-related diseases.
  • the MCL1 protein inhibitor has a significant therapeutic effect on Alzheimer's disease, so it can be used for the prevention and treatment of neurodegenerative diseases.
  • the present invention has been completed.
  • Mcl-1 myeloid cell leukemia-1) protein is one of the important anti-apoptotic protein members in the Bcl-2 (B-cell lymphoma-2) protein family, and it is overexpressed in a variety of tumor cells.
  • the overexpression of Mcl-1 protein is not only closely related to the occurrence and development of tumors, but also causes tumor cells to develop resistance to chemotherapeutic drugs. Therefore, small molecule inhibitors targeting Mcl-1 are the focus of future anti-tumor drug development. one.
  • MCL1 inhibitors can be used as mitochondrial autophagy inducers, and there are no drugs that target mitochondrial autophagy inducers or MCL1 protein inhibitors to treat inflammation and Alzheimer's disease.
  • the MCL1 protein inhibitor includes a compound having MCL1 protein inhibitory activity disclosed in patent document US2014/0235702A1.
  • the structure of the MCL1 protein inhibitor is shown in formula I:
  • R1 is X is halogen (such as fluorine, chlorine, bromine, iodine, preferably bromine);
  • R2 is R 3 is H, halogen, or hydroxyl.
  • the MCL1 protein inhibitor is compound UMI-77 or UMI-59:
  • the MCL1 protein inhibitors that can be used in the present invention include the compound itself or its derivatives.
  • the derivatives include polymorphs of the compound or pharmaceutically acceptable salts thereof, such as hydrochloride, sulfate, and phosphate. , Sulfonate, Carbonate, Acetate, Tartrate, Formate, Acetate or Isethionate, etc.
  • the preparation of these derivatives can usually be obtained by conventional technical means, and their effects and effects can be obtained according to cell experiments or equivalence experiments, which are all within the scope that those skilled in the art can understand and expect.
  • the MCL1 protein inhibitor or derivative thereof of the present invention can be directly used in the desired object, or mixed with a pharmaceutically acceptable carrier or prepared, and then administered in the form of a composition.
  • the dosage of the MCL1 protein inhibitor or derivative thereof that can be used in the present invention is usually 5-150 mg/kg/d, for example 50-120 mg/kg/d, preferably 80-90 mg/kg/d.
  • the administered dose is within the expected range of the clinician or laboratory staff. For example, the dose can be appropriately adjusted through effectiveness and safety tests to obtain the optimal dose.
  • the present invention provides an application of UMI-77 as a mitochondrial autophagy inducer in the preparation of drugs for the treatment of inflammation and neurodegenerative diseases.
  • the present invention discloses the application of UMI-77 as a mitochondrial autophagy inducer in the preparation of drugs for treating inflammation and neurodegenerative diseases.
  • UMI-77 induces mitochondria to enter lysosomes to cause mitochondrial autophagy.
  • the inflammation is acute hepatitis or inflammatory bowel disease.
  • the neurodegenerative disease is Alzheimer's disease.
  • the present invention also discloses a human embryonic kidney transformed cell HEK293Tmtkeima stable cell line, which was deposited in the China Type Culture Collection on March 7, 2019, with the preservation number: CCTCC NO: C201940.
  • the invention also discloses a method for constructing a stable cell line HEK293Tmtkeima transformed from human embryonic kidney, which comprises the following steps:
  • Lentiviral plasmid preparation After the mtkeima sequence is synthesized, it is connected to the lentiviral plasmid pCDH to form pCDH-mtkeima by restriction enzyme digestion, and the endotoxin-free plasmid pCDH-mtkeima is extracted, and the lentiviral packaging plasmids pMD2.0G and psPAX2 are extracted;
  • HEK293T cells were seeded in a 15cm culture dish at a density of 1*10 6 cells/ml, and three plasmids pCDH-mtkeima, pMD2.0G and psPAX2 were transfected 24 hours later, each plasmid was 10 ⁇ g; At 48 hours and 72 hours after staining, collect the supernatants, combine the supernatants and filter them with a 0.45 ⁇ m filter membrane, add 7.5ml virus concentrate solution to each 30ml filtered supernatant, add an appropriate amount of concentrated solution, and mix well.
  • HEK293T cells were planted in a 10cm culture dish at 5*10 5 cells/ml. After 24 hours, the prepared lentivirus was taken out from -80°C, fully dissolved, and dropped into the cell culture. Observe the infection rate after 48 hours. If the infection rate is greater than 90%, it is considered qualified. Add 1 ⁇ g/ml of puromycin to the qualified infected cell culture to remove uninfected cells. After 2-3 days, change the culture medium and expand the culture. The cells were cryopreserved, and the stable cell line HEK293Tmtkeima transformed into human embryonic kidney was obtained.
  • drug or composition refers to a composition to be administered for a specific purpose, including a pharmaceutical composition or a health care product composition.
  • active ingredient refers to an MCL1 protein inhibitor or derivative thereof.
  • administration method of the active ingredient or the pharmaceutical composition of the present invention is not particularly limited.
  • Representative administration methods include (but are not limited to): oral, intramuscular injection, intravenous injection, intravenous drip, enema, spray, external application, or intraperitoneal injection.
  • Solid dosage forms for oral administration include tablets, pills, powders, granules, or capsules.
  • the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectant, For example, glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, such as quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and g
  • Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of active ingredients or compounds in such compositions may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active ingredient can also be formed into a microcapsule form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the dosage forms of the compound of the present invention for topical administration include ointments, powders, patches, sprays and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
  • the compound of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage is the pharmaceutically effective dosage considered to be administered.
  • the daily dose administered is usually about 1 to 2000 mg, preferably about 6 to 600 mg.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skill range of a skilled physician.
  • the "safe and effective amount" refers to the amount of the compound that is sufficient to significantly improve the condition without causing serious side effects.
  • the present invention provides a new clinical application of MCL1 protein inhibitors, namely for inhibiting inflammation, preventing or treating inflammation-related diseases, and preventing or treating neurodegenerative diseases.
  • MCL1 protein inhibitors can significantly inhibit inflammation and show excellent therapeutic effects on neurodegenerative diseases.
  • the active ingredients of the present invention can also be used to induce mitochondrial autophagy in cultured cells, or to promote the interaction between MCL1 protein and LC3A protein.
  • concentration of the active ingredient of the present invention added is about 0.001 ⁇ M-1000 ⁇ M, preferably 0.01-100 ⁇ M. This concentration can be further adjusted and verified according to the desired effect. All are within the range conventionally obtained by those skilled in the art.
  • modulation includes treatment, prevention or interference.
  • treatment refers to the administration of the active ingredient of the present invention to a subject in need of treatment for the purpose of curing, relieving, improving, alleviating, or affecting the disease, symptom, and predisposition of the subject.
  • subject to treatment refers to mice, humans and other mammals.
  • inflammation refers to one or more of the following symptoms. Such as pain, fever, redness, swelling, temporary or permanent loss of function. Inflammation has many manifestations, including but not limited to, acute, viscous, atrophic, catarrhal, chronic, sclerotic, diffuse, diffuse, exudative, fibrous, fibrogenic, localized, granuloma, hyperplasia Sexuality, hypertrophy, interstitial, metastatic, necrotic, occlusive, substantial, plasticity, production, proliferation, pseudomembranous, purulent, sclerosing, serous fibrin, plasma, simple , Specificity, subacute, purulent, toxic, traumatic, and/or ulcerative.
  • inflammation also includes, but is not limited to, the following vascular appearances (polyarteritis, temporal arteritis); joint appearances (arthritis-crystalline, skeletal, psoriasis, reactive, rheumatic, and dependent characteristics); stomach Intestinal appearance (colitis); skin (dermatitis); or appearance of various tissues and organs (systemic lupus erythematosus).
  • vascular appearances polyarteritis, temporal arteritis
  • joint appearances arthritis-crystalline, skeletal, psoriasis, reactive, rheumatic, and dependent characteristics
  • stomach Intestinal appearance colitis
  • skin dermatitis
  • appearance of various tissues and organs systemic lupus erythematosus
  • the inflammation includes inflammation in a tissue selected from the group consisting of adipose tissue, liver tissue, muscle tissue, skin tissue, pancreas tissue, lung tissue, blood vessels, heart, bones, brain, spinal cord, retina, gastrointestinal tissue, reproduction System, or a combination.
  • inflammation-related diseases includes: multiple sclerosis, inflammatory bowel disease, arthritis, liver fibrosis, pulmonary fibrosis and the like.
  • neurodegenerative diseases includes: Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA) ), Pick disease, etc.
  • the present invention can obtain the following technical effects:
  • the present invention establishes a high-throughput screening model for mitochondrial autophagy.
  • mitochondrial autophagy Through the characteristics of mtkeima protein, mitochondrial autophagy can be conveniently indicated; the inducer UMI-77 that can gently induce mitochondrial autophagy is selected; UMI-77 is A specific inhibitor of MCL1 protein.
  • the MCL1 inhibitor UMI-77 of the present invention can induce mitochondrial autophagy.
  • the MCL1 inhibitor UMI-77 has a new function as an inducer of mitochondrial autophagy.
  • MCL1 inhibitors can be used as mitochondrial autophagy inducers.
  • the UMI-77 of the present invention has a significant inhibitory effect on inflammation; judging from the effect on acute hepatitis and inflammatory bowel disease mouse models, UMI-77 has a significant inhibitory effect on inflammation.
  • the UMI-77 of the present invention has a significant therapeutic effect on Alzheimer's disease; judging from the effect in the APP/PS1 mouse model, UMI-77 has a significant improvement effect on Alzheimer's disease.
  • the present invention utilizes Keima fluorescent protein and mitochondrial localization sequence to be fused to form mtKeima protein, and the human embryonic kidney transformed cell HEK293Tmtkeima stable cell line is constructed by this; the construction steps:
  • HEK293T cells were seeded in a 15cm culture dish at a density of 1*10 6 cells/ml, and three plasmids pCDH-mtkeima, pMD2.0G and psPAX2 were transfected 24 hours later, each plasmid was 10 ⁇ g.
  • At 48 hours and 72 hours after transfection collect the supernatants, combine the supernatants and filter them with a 0.45 ⁇ m filter membrane, add 7.5ml virus concentrated solution per 30ml filtered supernatant, add an appropriate amount of concentrated solution, and mix well. Mix it upside down every 30 minutes for 3-5 times for a total of 4 times. Place overnight at 4°C. Centrifuge at 4000g for 20 minutes at 4°C the next day, discard the supernatant, and resuspend the virus pellet with 1ml PBS (phosphate buffered saline). Store at -80°C.
  • PBS phosphate buffered saline
  • HEK293T cells are planted in a 10cm culture dish at 5*10 5 cells/ml. After 24 hours, the prepared lentivirus is taken out of the minus 80 degree refrigerator, fully dissolved and then dropped into the cell culture , Observe the infection rate after 48 hours, and the infection rate is greater than 90% as qualified. Add 1 ⁇ g/ml puromycin to the qualified infected cell culture to remove uninfected cells. After 2-3 days, change the medium and pass the cells, expand the culture, and freeze the cells to obtain the human embryonic kidney transformed cell HEK293Tmtkeima stable cell line. The cell line was deposited in the China Type Culture Collection on March 7, 2019, with the deposit number: CCTCC NO: C201940.
  • the excitation light of keima protein is 586nm under acidic conditions and 440nm under neutral conditions is used to indicate the occurrence of mitochondrial autophagy, and the high-throughput imaging system biotek cytation3 and software are used to analyze the number of (586nm positive cells/440nm total). Cell number) is an indicator for high-throughput quantitative analysis.
  • KH buffer and PBS are positive controls, which can induce autophagy (including mitochondrial autophagy).
  • KH buffer and PBS phosphate buffer
  • the fluorescence excited by 586nm increases significantly.
  • NC untreated did not increase significantly.
  • UMI-77 induces mitochondrial autophagy (mtkeima signal).
  • the bar graph represents the degree of mitochondrial autophagy induction. The higher the induction, the stronger the induction.
  • HEK293T cells are seeded at a density of 2*10 5 cells/ml
  • Hela cells and U2OS cells are seeded in a 6-well plate at a density of 1.5*10 5 cells/ml, 2 ml per well. Each cell has 6 wells.
  • UMI-77 was added at 0 hours, 3 hours, 6 hours, and 9 hours respectively, and the final concentration was 5 ⁇ M.
  • Collect samples with 250 ⁇ l 2XSDS loading buffer at 12 hours. Heat at 100°C for 10 minutes.
  • Immunohybridization load 10 ⁇ l of each sample, electrophoresis 90V2h; transfer membrane 300mA, 1h.
  • 5% skim milk was blocked at room temperature for 1 hour, and antibody (Tom20 antibody was purchased from Cell Signaling Technology, #42406S; Tim23 antibody was purchased from Proteintech, #11123-1-AP; Tubulin antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd., #M1305-2; Calnexin antibody was purchased from Cell Signaling Technology, #2433S) Incubate overnight at 4 degrees at a certain dilution ratio (1:5000 for Tubulin antibody, and 1:1000 for the rest), and wash 3 times each time with PBST (phosphate buffer saline + 0.1% Tween20) 10 minutes.
  • PBST phosphate buffer saline + 0.1% Tween20
  • Goat anti-Mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31430; Goat anti-Rabbit IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31460) diluted 1: 20000 Incubate for 1 hour than room temperature, wash 3 times with PBST for 10 minutes each time. ECL color development.
  • U2OS cells are seeded in a 6-well plate at a density of 1.5*10 5 cells/ml, 2 ml per well, 5 wells. After 24 hours, UMI-77 was added at 0 hours, 12 hours, 24 hours, and 36 hours, and the final concentration was 7.5 ⁇ M. Collect samples with 250 ⁇ l 2XSDS loading buffer at 48 hours. Add staurosporine to a final concentration of 1 ⁇ M 3 hours before the collection. Heat at 100 degrees for 10 minutes. Immunohybridization: load 10 ⁇ l of each sample, electrophoresis 90V2h; transfer membrane 300mA, 1h.
  • Goat anti-Mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31430; Goat anti-Rabbit IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31460) diluted 1: 20000 Incubate for 1 hour than room temperature, wash 3 times with PBST for 10 minutes each time. ECL color development.
  • HEK293T cells are seeded in a 6-well plate at a density of 2*10 5 cells/ml, 2 ml per well, 5 wells. Hela cells were seeded in a 6-well plate at a density of 1.5*10 5 cells/ml, 2 ml per well, 5 wells. After 24 hours, UMI-77 was added at a final concentration of 5 ⁇ M and treated for 24 hours. MG132 2 ⁇ M, E64D 2 ⁇ M and ammonium chloride 20mM, Leupeptin (leupeptin) 50 ⁇ M were added 12 hours before sampling. Collect samples with 250 ⁇ l 2XSDS loading buffer in 24 hours. Heat at 100 degrees for 10 minutes.
  • Immunohybridization Load 10 ⁇ l of each sample, electrophoresis at 90V for 2h; transfer membrane to 300mA, 1h. Blocked with 5% skimmed milk for 1h, antibody (Tom20 antibody was purchased from Cell Signaling Technology, #42406S; Tim23 antibody was purchased from Proteintech, #11123-1-AP; Tubulin antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd., #M1305-2) A certain dilution ratio (1:5000 for Tubulin antibody, 1:1000 for the rest) was incubated overnight at 4 degrees, and washed with PBST 3 times for 10 minutes each time.
  • Goat anti-Mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31430; Goat anti-Rabbit IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31460) diluted 1: 20000 After incubating for 1 hour than room temperature, washing with PBST 3 times for 10 minutes each time, ECL develops color.
  • HEK293T cells are seeded in a 6-well plate at a density of 2*105 cells/ml, 2ml per well, 5 wells. After 24 hours, add UMI-77 to a final concentration of 5 ⁇ M and treat for 24 hours. Add E64D 2 ⁇ M, ammonium chloride 20mM, and Leupeptin (leupeptin) 50 ⁇ M 12 hours before sampling. After being fixed with glutaraldehyde, the samples were prepared and tested by electron microscope.
  • the screened effective mitochondrial autophagy inducer UMI-77 has the following characteristics:
  • Mitochondrial degradation passes through the lysosomal pathway.
  • adding lysosomal inhibitor E64D or ammonium chloride and Leupeptin (leupeptin) can prevent the degradation of mitochondrial marker proteins Tom20 and Tim23;
  • Mitochondria will be wrapped by autophagy lysosome membrane. If observed with electron microscope, it can be seen that multiple mitochondria are wrapped by autophagy lysosome.
  • mice with C57BL/6 genetic background were purchased from Nanjing Model Animal Center. Divided into 4 groups, each with 6 animals, and the experiment was carried out after normal feeding for 1 week. All drugs are dissolved in a solvent of 2% DMSO + 30% PEG400 + water. Weigh each mouse and inject 200 ⁇ l of the drug into the intraperitoneal cavity of UMI-77 30mg/kg and Nec1 2.5mg/kg according to the body weight. The LPS induction group was intraperitoneally injected with 200 ⁇ l solvent. The NC group does not do processing.
  • LPS/D-Gal lipopolysaccharide/D-galactosamine concentration
  • D-Gal 400 mg/kg
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • UMI-77 can significantly inhibit liver damage.
  • Nec1 is a known RIPK1 protein inhibitor, and UMI-77 is more effective than Nec1.
  • UMI-77 can significantly inhibit liver injury. UMI-77 restores the integrity of liver cells.
  • Example 4 UMI-77 induces mitochondrial autophagy by inducing the interaction between MCL1 and LC3A (autophagy marker protein)
  • HEK293T cells are seeded at a density of 2*10 5 cells/ml, and Hela cells are seeded in a 6-well plate at a density of 1.5*10 5 cells/ml, 2 ml per well. Each cell has 6 wells.
  • Transfection method pre-mix 50 ⁇ l serum-free DMEM medium and 1 ⁇ g DNA, 50 ⁇ l serum-free DMEM medium and 2 ⁇ l lip2000. Finally, the two components were mixed, allowed to stand at room temperature for 20 minutes, and dropped into the culture medium.
  • 5% skim milk was blocked at room temperature for 1 hour, and the antibody (Tom20 antibody was purchased from Cell Signaling Technology, #42406S; Tim23 antibody was purchased from Proteintech, #11123-1-AP; Tubulin antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd., #M1305-2, MCL1 antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd., #ET1606-14) and incubated at a certain dilution ratio (1:5000 for Tubulin antibody, and 1:1000 for the rest) at 4 degrees overnight, PBST (phosphate buffer saline + 0.1% Tween 20) ) Wash 3 times for 10 minutes each time.
  • PBST phosphate buffer saline + 0.1% Tween 20
  • Goat anti-Mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31430; Goat anti-Rabbit IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31460) diluted 1: 20000 Incubate for 1 hour than room temperature, wash 3 times with PBST for 10 minutes each time. ECL color development.
  • HEK293T cells were seeded in a 12-well plate at 2*10 5 cells/ml, 1 ml per well, and plated with immunofluorescence slides in advance. After 24 hours, add UMI-77 10 ⁇ M to treat for 5 hours, remove the medium, wash twice with PBS, and add an appropriate amount of 4% paraformaldehyde for fixation for 20 minutes. Wash twice with PBS, use Blocking solution in the In Situ Red Starter Kit Mouse/Rabbit kit (purchased from Sigma-Aldrich, #DUO92101) was blocked for 60 minutes at 37 degrees.
  • the antibody diluent in the above kit to dilute the antibody (MCL1 antibody purchased from Santa cruz, #sc-69840, 1:200 dilution), LC3A antibody purchased from Abcam, #ab62720, 1:500 dilution), incubate overnight at 4 degrees . Wash the film twice with the washing reagent A in the kit, each time for 10 minutes. Incubate the secondary antibodies Anti-rabbit-PLUS and Anti-mouse-MINUS according to the instructions of the kit at 37 degrees for 60 minutes. After that, wash the film twice with washing reagent A, and ligase for 30 minutes at 37 degrees according to the instructions of the kit.
  • Experimental method HEK293T cells were seeded in a 10cm petri dish at 2*10 5 cells/ml. After 24 hours, pCMV3-MCL1-3xFlag and pCDNA3.1-HA-LC3A or pCDNA3.1-HA-LC3B plasmids were co-transfected, 4 ⁇ g each.
  • Transfection method pre-mix 500 ⁇ l serum-free DMEM medium and 8 ⁇ g DNA, 500 ⁇ l serum-free DMEM medium and 16 ⁇ l PEI (polyethyleneimine) transfection reagent. Finally, the two components were mixed, allowed to stand at room temperature for 20 minutes, and dropped into the culture medium.
  • PEI polyethyleneimine
  • HEK293T cells were seeded in a 10cm petri dish at 2*10 5 cells/ml. After 24 hours, pCMV3-MCL1-3xFlag and mutants and pCDNA3.1-HA-LC3A plasmids were co-transfected, 4 ⁇ g each.
  • Transfection method pre-mix 500 ⁇ l serum-free DMEM medium and 8 ⁇ g DNA, 500 ⁇ l serum-free DMEM medium and 16 ⁇ l PEI (polyethyleneimine) transfection reagent. Finally, the two components were mixed, allowed to stand at room temperature for 20 minutes, and dropped into the culture medium. After 24 hours of transfection, UMI-77 10 ⁇ M was added for 5 hours, and the cells were washed twice with PBS.
  • the remaining prepared cell lysis supernatant was mixed with the pretreated agarose gel coupled with Flag antibody, and mixed overnight at 4 degrees. The next day, the agarose gel was collected by centrifugation at 2000 rpm for 3 minutes, and washed with RIPA 3 times, and centrifuged at 2000 rpm for 3 minutes each time. Finally, collect the agarose gel and resuspend it in 30 ⁇ l of 2X SDS loading buffer, and boil it at 100°C for 10 minutes. Immunohybridization: Load 10 ⁇ l of each sample, electrophoresis at 90V for 2h; transfer membrane to 300mA, 1h.
  • the two LIR motifs contained in MCL1 were mutated separately.
  • the first LIR motif mutation has four forms: W261A, I264A, W261A/I264A, ⁇ LIR.
  • the second LIR motif mutation has three forms: F318A, V321A, F318A/V321A.
  • the first LIR motif mutation significantly affected the interaction between MCL1 and LC3A.
  • the second LIR motif has almost no effect.
  • mice 7-8 weeks old male wild-type mice with C57BL/6 genetic background freely drink 2% DSS solution (the solution needs to be freshly prepared) for 6 days, then change to normal water until the end of the experiment.
  • UMI77 5mg/kg body weight
  • the weight change, stool shape and blood in the stool were recorded every day.
  • the histopathology score is divided into inflammatory cell infiltration score and tissue damage score refer to Immunity.2010; 32(3) :379-91, finally calculate the disease activity index.
  • UMI77 can significantly slow down weight loss, reduce disease activity index, restore the normal state of intestinal tissue and its cells, and reduce the expression of inflammatory factors, suggesting that it can protect inflammatory bowel disease.
  • Example 6 restores the spatial memory and learning ability of APP/PS1 model mice
  • APP/PS1 mice One of the commonly used mouse models of Alzheimer's disease is APP/PS1 mice.
  • APP/PS1 mice with genetic background of C57BL/6 and control mice were purchased from Nanjing Model Animal Center. Divided into three groups, the control group, the APP/PS1 model group, and the APP/PS1 model administration group, each with 5-7 animals.
  • APP/PS1 began intraperitoneal injection of UMI-77 13mg/kg at the age of 4 months. The drug was administered every other day for 4 months, after which a water maze experiment was performed.
  • the experiment is mainly divided into two parts: positioning cruise experiment and space search experiment.
  • the positioning cruise experiment is used to measure the learning and memory ability of mice in the water maze.
  • the spatial search experiment measures the ability of mice to retain memory of the platform's spatial location.
  • Positioning cruise experiment Divide the pool containing titanium dioxide water into 4 quadrants (northwest, northeast, southwest, southeast), and place the platform about 1cm underwater in the southeast quadrant. Put the mouse head towards the pool wall and put it into the water 4 times in the order of northwest, northeast, southeast and southwest. Record each time the mouse finds the underwater platform. If the time exceeds 60 seconds, guide the mouse to the platform, let it stay for 20 seconds, and record the time to find the platform as 60 seconds. Each mouse was trained 4 times a day for a total of 4 days, and the time to find the platform (escape latency) was recorded.
  • Space search experiment 24 hours after the positioning cruise experiment is over, the platform will be removed and the space search experiment will start for 60 seconds. Place the mouse into the water from the opposite side of the original platform quadrant, and record the number of times the mouse passes through the original platform position and the movement track.
  • Alzheimer's disease is a serious neurodegenerative disease, and currently there is no specific treatment. In previous studies, it was found that there is a large amount of amyloid accumulation and damaged mitochondria accumulation in the brain of Alzheimer's patients.
  • the currently known clinical drugs are mainly aimed at the clearance and degradation of amyloid.
  • UMI-77 can induce mitochondrial autophagy and help eliminate damaged mitochondria, indicating its potential for the treatment of Alzheimer's disease.

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Abstract

MCL1蛋白抑制剂在制备治疗炎症及神经退行性疾病药物中的应用。提出了MCL1抑制剂UMI-77对线粒体自噬的诱导作用，通过对急性肝炎，炎症性肠病和APP/PS1小鼠模型中的效果来看，UMI-77对炎症有显著的抑制效果，对阿尔兹海默症有显著缓解效果。

Description

MCL1蛋白抑制剂在制备治疗炎症及神经退行性疾病药物中的应用 技术领域
本发明属于炎症治疗技术领域，具体地说，涉及一种UMI-77及其衍生物作为线粒体自噬诱导剂在制备治疗炎症及神经退行性疾病药物中的应用。
背景技术
线粒体自噬在炎症的抑制过程中发挥重要作用，而炎症是机体对抗感染的基本策略，同时也是导致疾病加重的主要原因，如某些神经退行性疾病，阿尔兹海默症。因此，筛选新的安全有效的线粒体自噬诱导剂是可能的抑制炎症，缓解疾病的治疗策略。现有的线粒体自噬诱导剂主要原理为破坏线粒体的功能，使细胞被迫发生线粒体自噬。虽然可以诱导线粒体自噬，但由于具有较大的毒性，并不能在临床应用中发挥作用。
目前并没有发现MCL1抑制剂可以作为线粒体自噬诱导剂，也没有以线粒体自噬诱导剂或者MCL1蛋白抑制剂为靶点的药物治疗炎症和阿尔兹海默症。
发明内容
本发明的目的在于提供一种线粒体自噬诱导剂及其应用。
本发明的第一方面，提供了一种MCL1蛋白抑制剂的用途，用于制备药物或组合物，所述药物或组合物用于选自下组的一种或多种用途：
(1)诱导线粒体自噬；
(2)预防或抑制炎症；
(3)预防或治疗炎症相关疾病；和
(4)预防或治疗神经退行性疾病。
另一优选例中，所述炎症包括选自下组的组织中的炎症：脂肪组织、肾脏组织、肝脏组织、肌肉组织、皮肤组织、胰腺组织、肺组织、血管、心脏、骨骼、大脑、脊髓、视网膜、胃肠道组织、生殖系统，或其组合。
在另一优选例中，所述炎症包括肝炎(如急性肝炎)、肠炎(如溃疡性肠炎)、自身免疫疾病(如系统性红斑狼疮、类风湿性关节炎)、代谢疾病炎症(如糖尿病引起的肾炎)。
在另一优选例中，所述神经退行性疾病包括阿尔茨海默病(AD)、帕金森病(PD)、亨廷顿病(HD)、肌萎缩性侧索硬化(ALS)、多发性硬化症(MS)、不同类型脊髓小脑共济失调(SCA)、Pick病。
在另一优选例中，所述MCL1蛋白抑制剂包括专利文献US2014/0235702A1中公开的具有MCL1蛋白抑制活性的化合物。
在另一优选例中，所述MCL1蛋白抑制剂为结构如式I所示的化合物或其药学可接受的盐：
Figure PCTCN2020083442-appb-000001
其中，
R1为
Figure PCTCN2020083442-appb-000002
X为卤素；
R2为
Figure PCTCN2020083442-appb-000003
R 3为H、卤素、或羟基。
在另一优选例中，所述MCL1蛋白抑制剂为化合物UMI-77或UMI-59：
Figure PCTCN2020083442-appb-000004
Figure PCTCN2020083442-appb-000005
在另一优选例中，所述药物或组合物含有安全有效量的MCL1蛋白抑制剂，和药学上可接受的载体。
在另一优选例中，所述药物或组合物还包括抗氧化剂。
在另一优选例中，所述药物或组合物为单元剂型。
在另一优选例中，所述药物或组合物包括胃肠给药剂型或胃肠外给药剂型。
在另一优选例中，所述药物或组合物包括片剂、丸剂、散剂、颗粒剂、胶囊剂、糖浆、乳剂、悬浮剂、注射剂、贴剂、滴剂、膏剂、栓剂或喷雾剂。
在另一优选例中，所述药物或组合物的施用方式包括口服、肌肉注射、静脉注射、静脉滴注、喷雾、或腹腔注射。
在另一优选例中，所述安全有效量指的是5-1000mg/kg；优选的为50-1000mg/kg。
在另一优选例中，所述的安全有效量包括50-200mg/kg(小鼠)，或500-1000mg/kg(人)。
在另一优选例中，所述的安全有效量为100-700mg/kg，更优选地为500-700mg/kg。
本发明的第二方面，提供了一种诱导线粒体自噬的方法，包括步骤：给需要的对象(如体外培养的细胞)施用MCL1蛋白抑制剂。所述MCL1蛋白抑制剂如上所述。
在另一优选例中，所述方法为体外非治疗性的方法。
在另一优选例中，所述MCL1蛋白抑制剂的浓度为约0.001μM-1000μM，优选地为约0.1uM-100μM。
本发明的第三方面，提供了一种促进MCL1蛋白和LC3A蛋白相互作用的方法，其中包括步骤：在包含MCL1蛋白和LC3A蛋白的体系中，施加MCL1蛋白抑制剂。所述MCL1蛋白抑制剂如上所述。
在另一优选例中，所述包含MCL1蛋白和LC3A蛋白的体系为细胞培养 液。
本发明的第四方面，提供了一种抑制炎症或治疗炎症相关疾病的组合物，所述组合物包括MCL1蛋白抑制剂。所述MCL1蛋白抑制剂如上所述。
在另一优选例中，所述组合物还包括药学上可接受的载体。
在另一优选例中，所述组合物还包括抗氧化剂。
本发明的第五方面，提供了一种预防或治疗神经退行性疾病的方法，所述方法包括步骤：给需要的对象施用安全有效量的MCL1蛋白抑制剂。所述MCL1蛋白抑制剂如上所述。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本发明的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1是本发明验证人胚肾转化细胞HEK293Tmtkeima系统是否可以指征线粒体自噬；
图2是本发明对2000多个FDA批准的药进行高通量筛选；颜色深浅代表586nm激发荧光与阴性对照对比的强弱；颜色越深代表线粒体自噬诱导越强；
图3是本发明33个阳性药物二次验证，柱形图代表线粒体自噬诱导程度高低，越高代表诱导越强；实线是阴性对照基准线，虚线是1.5倍上升线；
图4是本发明UMI-77诱导线粒体标记蛋白Tom20和Tim23降解；
图5是本发明UMI-77处理细胞免疫杂交(western blot)检测Caspase 3的结果；
图6是本发明人胚肾转化细胞HEK293Tmtkeima系统在UMI-77处理下诱导线粒体自噬结果；图像为biotek cytation3拍摄,放大倍数4X；红色(EM586对应的上下两幅图)为mtkeima586nm激发荧光，代表发生线粒体自噬的线 粒体。绿色(EM469对应的上下两幅图)为488nm激发荧光代表正常线粒体；
图7是本发明人胚肾转化细胞HEK293Tmtkeima细胞用UMI-77处理后活细胞拍摄激光共聚焦图片观察线粒体和溶酶体共定位；
图8是本发明UMI-77处理HEK293T和Hela细胞导致的线粒体通过溶酶体发生降解；
图9是本发明透射电子显微镜检测UMI-77处理HEK293T细胞后线粒体的结果；
图10是本发明小鼠血液检测谷丙转氨酶和谷草转氨酶的活力测定结果，UMI-77抑制肝细胞破损；
图11是本发明HE(苏木精-伊红)染色小鼠肝脏切片显示UMI-77恢复肝细胞正常形态；
图12是本发明UMI-77诱导线粒体降解需要MCL1蛋白；
图13是本发明
Figure PCTCN2020083442-appb-000006
In Situ Red Starter Kit Mouse/Rabbit试剂盒(购自Sigma-Aldrich，#DUO92101)检测MCL1和LC3A相互作用；
图14是本发明免疫共沉淀实验显示UMI-77诱导LC3A和MCL1相互作用增强；
图15是本发明免疫共沉淀显示MCL1与LC3A相互作用通过LIR motif实现；
图16是本发明小鼠炎症性肠病模型及UMI77处理实验流程图；
图17是本发明小鼠体重统计结果，在DSS诱导小鼠炎症性肠病模型中，UMI77可以显著性减缓体重下降幅度，提示其可以保护炎症性肠病；
图18是本发明小鼠疾病活动指数统计结果，疾病活动指数基于体重变化、小鼠便血和粪便性状计算获得；
图19是本发明小鼠结直肠代表性图，小鼠结直肠长度统计结果；
图20是本发明小鼠结直肠HE染色图，小鼠结直肠组织病理评分；
图21是本发明小鼠结直肠组织中炎症因子表达水平；
图22是本发明定位巡航实验记录每组小鼠的逃避潜伏期；
图23是本发明空间搜索实验记录小鼠穿过原平台的次数；
图24是本发明空间搜索实验记录有代表性小鼠的运动轨迹。
具体实施方式
本发明人通过广泛而深入的研究，意外发现MCL1蛋白抑制剂能够诱导线粒体自噬，并促进MCL1蛋白和LC3A蛋白相互作用。实验结果表明，MCL1蛋白抑制剂对炎症有显著的抑制效果，进而能够用于炎症相关疾病的预防或治疗。而且发现，MCL1蛋白抑制剂对阿尔兹海默症的治疗效果显著，因此能够用于神经退行性疾病的预防和治疗。在此基础上，完成了本发明。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
Mcl-1及其抑制剂
Mcl-1(myeloid cell leukemia-1)蛋白是Bcl-2(B-cell lymphoma-2)蛋白家族中重要的抗凋亡蛋白成员之一,在多种肿瘤细胞中过表达。Mcl-1蛋白的过表达不仅与肿瘤的发生发展密切相关,还会导致肿瘤细胞对化疗药物产生耐药性,因此以Mcl-1为靶点的小分子抑制剂是未来抗肿瘤药物研发的重点之一。
但是目前并没有发现MCL1抑制剂可以作为线粒体自噬诱导剂，也没有以线粒体自噬诱导剂或者MCL1蛋白抑制剂为靶点的药物治疗炎症和阿尔兹海默症。
在本发明优选地实施方式中，所述MCL1蛋白抑制剂包括专利文献US2014/0235702A1中公开的具有MCL1蛋白抑制活性的化合物。
在另一优选例中，所述MCL1蛋白抑制剂的结构如式I所示：
Figure PCTCN2020083442-appb-000007
其中，
R1为
Figure PCTCN2020083442-appb-000008
X为卤素(如氟、氯、溴、碘，优选为溴)；
R2为
Figure PCTCN2020083442-appb-000009
R 3为H、卤素、或羟基。
在另一优选例中，所述MCL1蛋白抑制剂为化合物UMI-77或UMI-59：
Figure PCTCN2020083442-appb-000010
可用于本发明的MCL1蛋白抑制剂包括化合物本身或其衍生物，所述的衍生物的例子有化合物的多晶型、或其药学上可接受的盐，例如盐酸盐、 硫酸盐、磷酸盐、磺酸盐、碳酸盐、醋酸盐、酒石酸盐、甲酸盐、乙酸盐或羟乙基磺酸盐等。这些衍生物的制备通常可以通过常规技术手段获得，且其作用和效果可以根据细胞实验或等效性实验测试获得，均在本领域技术人员可以理解和预期的范围内。
本发明MCL1蛋白抑制剂或其衍生物可以直接用于所需的对象，或与药学上可接受的载体混合或经制备后，以组合物的形式进行施用。可用于本发明的MCL1蛋白抑制剂或其衍生物的施用剂量通常为5-150mg/kg/d，例如50-120mg/kg/d，优选为80-90mg/kg/d。而该施用的剂量在临床医师或实验室人员的可预期范围内，例如可通过有效性、安全性测试，对其剂量进行适当的调整，从而获得最优的施用剂量。
在本发明的一个优选地实施方式中，本发明提供了一种UMI-77作为线粒体自噬诱导剂在制备治疗炎症和神经退行性疾病药物中的应用。
具体地，本发明公开了一种UMI-77作为线粒体自噬诱导剂在制备治疗炎症及神经退行性疾病药物中的应用。
可选地，UMI-77诱导线粒体进入溶酶体发生线粒体自噬。
可选地，炎症为急性肝炎或者炎症性肠病。
可选地，神经退行性疾病为阿尔兹海默症。
本发明还公开了一种人胚肾转化细胞HEK293Tmtkeima稳定细胞株，该细胞株于2019年3月7日保藏于中国典型培养物保藏中心，保藏号为：CCTCC NO:C201940。
本发明还公开了一种人胚肾转化细胞HEK293Tmtkeima稳定细胞株的构建方法，包括以下步骤：
1)慢病毒质粒准备：mtkeima序列合成后通过酶切连接方法，连入慢病毒质粒pCDH中形成pCDH-mtkeima，抽提无内毒素的质粒pCDH-mtkeima，慢病毒包装质粒pMD2.0G和psPAX2；
2)慢病毒制备：HEK293T细胞以1*10 6个/ml密度种于15cm培养皿中，24小时后转染三个质粒pCDH-mtkeima，pMD2.0G和psPAX2，每个质粒10μg；分别在转染后48小时和72小时，收集上清，合并上清并用0.45μm滤膜过滤，以每30ml过滤上清加入7.5ml病毒浓缩溶液的比例，加入适量的浓缩溶液，混匀后，以每隔30分钟颠倒混匀3-5次，共进行4次；4℃放置过夜，第二天4000g，4℃离心20分钟，倒掉上清，用1ml PBS重悬病毒沉 淀，-80℃保存；
3)慢病毒感染：HEK293T细胞以5*10 5个/ml种于10cm培养皿中，24小时后，将制作好的慢病毒从-80℃中取出，充分溶解后滴入细胞培养物中，48小时后观察感染率，感染率大于90％评价为合格，向合格的感染细胞培养物中加入1μg/ml的嘌呤霉素清除未感染的细胞，2-3天后换液传代细胞，扩大培养，冻存细胞，得到人胚肾转化细胞HEK293Tmtkeima稳定细胞株。
药物或组合物
如本文所用，术语“药物或组合物”是指将用于特定目的而被施用的组合物，包括药物组合物或保健品组合物。
如本文所用，术语“活性成分”指的是MCL1蛋白抑制剂或其衍生物。
本发明活性成分或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、肌肉注射、静脉注射、静脉滴注、灌肠、喷雾、外敷、或腹腔注射。
用于口服给药的固体剂型包括片剂、丸剂、散剂、颗粒剂、或胶囊剂。在这些固体剂型中，活性成分与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性成分或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性成分也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳剂、溶液、悬浮剂、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬 浮剂、甜味剂、矫味剂和香料。
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷雾剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的化合物联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为约1～2000mg，优选约6～600mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。通常其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。
应用
本发明提供了MCL1蛋白抑制剂的临床新应用，即用于抑制炎症、预防或治疗炎症相关疾病，预防或治疗神经退行性疾病。本发明人发现MCL1蛋白抑制剂能够显著抑制炎症，并且对神经退行性疾病表现出了优异的治疗效果。
而用于细胞实验时，本发明活性成分还可以用于诱导培养中的细胞的诱导线粒体自噬，或者促进MCL1蛋白和LC3A蛋白相互作用。通常，培养的细胞中，所添加的本发明活性成分的浓度大约为0.001μM-1000μM，优选地为0.01-100μM，该浓度可以根据所需达到的效果对添加的浓度进行进一步调整和验证，这均在本领域技术人员可以常规获得的范围内。
本发明中，术语“调节”包括治疗、预防或干扰。
术语“治疗”是指基于治愈、缓解、改善、减轻、影响治疗对象疾病、症状、疾病体质(predisposition)的目的而给予需要治疗的对象本发明的活性成分。
术语“治疗对象”是指鼠、人及其他哺乳动物。
术语“炎症”指有一种或多种以下病症特征。如疼痛、发热、发红、肿大、暂时性或永久性功能缺失。炎症有很多表现形式，包括但不限于，急性、 粘性、萎缩性、卡他性、慢性、硬化、弥漫性、散布性、渗出性、含纤维性、成纤维性、局部、肉芽肿、增生性、肥大性、间质性、转移性、坏死性、闭塞性、实质性、可塑性、产生性、增殖性、假膜性、化脓性、致硬化性、浆液纤维蛋白性、血浆性、单纯性、特异性、亚急性、催脓性、毒性、外伤性、和/或溃疡性。更进一步的，炎症还包括，但不限于以下血管表象(多动脉炎，颞动脉炎)；关节表象(关节炎——结晶性、骨骼性、牛皮癣、反应性、风湿性、赖特性)；胃肠道表象(结肠炎)；皮肤(皮炎)；或多种组织和器官的表象(全身性红斑狼疮)。
所述炎症包括选自下组的组织中的炎症：脂肪组织、肝脏组织、肌肉组织、皮肤组织、胰腺组织、肺组织、血管、心脏、骨骼、大脑、脊髓、视网膜、胃肠道组织、生殖系统，或其组合。
术语“炎症相关疾病”包括:多发性硬化症、炎症性肠病、关节炎、肝纤维化、肺纤维化等。
术语“神经退行性疾病”包括：阿尔茨海默病(AD)、帕金森病(PD)、亨廷顿病(HD)、肌萎缩性侧索硬化(ALS)、不同类型脊髓小脑共济失调(SCA)、Pick病等。
与现有技术相比，本发明可以获得包括以下技术效果：
1)本发明通过建立线粒体自噬的高通量筛选模型，通过mtkeima蛋白的特性，可以方便的指征线粒体自噬；筛选到可以温和诱导线粒体自噬的诱导剂UMI-77；UMI-77为MCL1蛋白的特异性抑制剂。
2)本发明MCL1抑制剂UMI-77对线粒体自噬的诱导作用，MCL1抑制剂UMI-77作为线粒体自噬诱导剂的新功能。同时MCL1抑制剂都可以作为线粒体自噬诱导剂的可能性。
3)本发明UMI-77对与炎症的显著抑制效果；通过对急性肝炎和炎症性肠病小鼠模型中的效果来看，UMI-77对炎症有显著的抑制效果。
4)本发明UMI-77对阿尔兹海默症的治疗效果显著；通过对APP/PS1小鼠模型中的效果来看，UMI-77对阿尔兹海默症有显著的改善效果。
当然，实施本发明的任一产品并不一定需要同时达到以上所述的所有技术效果。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂 商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1线粒体自噬的高通量筛选模型的建立：
本发明利用Keima荧光蛋白和线粒体定位序列融合成mtKeima蛋白，并以此构建人胚肾转化细胞HEK293Tmtkeima稳定细胞株；构建步骤：
1)慢病毒质粒准备：mtkeima序列根据北京博尔迈生物技术有限公司(MBL BEIJING BIOTECH CO.,LTD)公司质粒pMitophagy Keima-Red mPark2(Hyg)中提供的MT-mKeima-Red序列全基因合成后通过酶切连接方法，连入慢病毒质粒pCDH中形成pCDH-mtkeima，抽提无内毒素的质粒pCDH-mtkeima，慢病毒包装质粒pMD2.0G和psPAX2。
2)慢病毒制备：HEK293T细胞以1*10 6个/ml密度种于15cm培养皿中，24小时后转染三个质粒pCDH-mtkeima，pMD2.0G和psPAX2，每个质粒10μg。分别在转染后48小时和72小时，收集上清，合并上清并用0.45μm滤膜过滤，以每30ml过滤上清加入7.5ml病毒浓缩溶液的比例，加入适量的浓缩溶液，混匀后，以每隔30分钟颠倒混匀3-5次，共进行4次。4℃放置过夜。第二天4000g，4℃离心20分钟，倒掉上清，用1ml PBS(磷酸缓冲液)重悬病毒沉淀。-80℃保存。
3)慢病毒感染：HEK293T细胞以5*10 5个/ml种于10cm培养皿中，24小时后，将制作好的慢病毒从负80度冰箱中取出，充分溶解后滴入细胞培养物中，48小时后观察感染率，感染率大于90％评价为合格。向合格的感染细胞培养物中加入1μg/ml的嘌呤霉素清除未感染的细胞，2-3天后换液传代细胞，扩大培养，冻存细胞，得到人胚肾转化细胞HEK293Tmtkeima稳定细胞株，其细胞株于2019年3月7日保藏于中国典型培养物保藏中心，保藏号为：CCTCC NO：C201940。
利用keima蛋白在酸性条件下激发光为586nm，中性条件下激发光为440nm指征线粒体自噬的发生，并利用高通量成像系统biotek cytation3和软件分析，以(586nm阳性细胞数/440nm全细胞数)为指标进行高通量定量分析。
1、实验方法：人胚肾转化细胞HEK293Tmtkeima细胞以1.5*10 5个/ml种于96孔黑色酶标板中，每孔种100μl，24小时后替换培养基为KH buffer和PBS，每个处理3个重复，于37℃，5％CO 2培养条件下，处理4小时后， 直接于biotek cytation3拍照。以469nm激发光做对焦通道，每孔拍摄4张图片，利用仪器软件处理图像。
如图1所示，KH buffer和PBS为阳性对照，可以诱导自噬(包括线粒体自噬)，KH buffer(KH缓冲液)和PBS(磷酸缓冲液)处理细胞后，586nm激发的荧光明显上升，而NC(未作处理的)没有明显上升。
2、实验方法：人胚肾转化细胞HEK293Tmtkeima细胞以1.5*10 5个/ml种于96孔黑色酶标板中，每孔种100μl。24小时后替换阳性对照孔培养基为KHbuffer和PBS，每个处理3个重复。用安捷伦bravo自动化液体处理平台高通量加入FDA批准的药物，每孔加入10μM的药物，于37℃，5％CO 2培养条件下，处理6小时后，直接于biotek cytation3拍照。以469nm激发光做对焦通道，每孔拍摄4张图片，利用仪器软件处理图像。并以(586nm阳性细胞数/469nm全细胞数)为指标进行统计分析，Graphpad处理数据以热图展示。
如图2所示，对2000多个FDA批准的药进行高通量筛选，图像处理后，以(586nm阳性细胞数/440nm全细胞数)为指标进行统计分析，以热图方式展示。红色深浅代表586nm激发荧光与阴性对照对比的强弱。红色越强代表线粒体自噬诱导越强。
如图3所示，UMI-77诱导线粒体自噬(mtkeima信号)，柱形图代表线粒体自噬诱导程度高低，越高代表诱导越强。
实施例2有效线粒体自噬诱导剂UMI-77的确证：
1、实验方法：HEK293T细胞以2*10 5个/ml，Hela细胞和U2OS细胞以1.5*10 5个/ml的密度种于6孔板中，每孔2ml。每个细胞种6个孔。24小时后，分别在0小时，3小时，6小时，9小时加入UMI-77，终浓度为5μM。在12小时统一用250μl 2XSDS上样缓冲液收样。100℃加热10分钟。免疫杂交：每个样品上样10μl，电泳90V2h；转膜300mA，1h。5％脱脂牛奶室温封闭1h，抗体(Tom20抗体购自Cell Signaling Technology，#42406S；Tim23抗体购自Proteintech，#11123-1-AP；Tubulin抗体购自杭州华安生物技术有限公司，#M1305-2；Calnexin抗体购自Cell Signaling Technology，#2433S)以一定稀释比(Tubulin抗体为1：5000，其余为1：1000)4度孵育过夜，PBST(磷酸盐缓冲液+0.1％Tween20)洗涤3遍每次10分钟。二抗(Goat anti-Mouse  IgG(H+L)二抗购自Thermo Fisher Scientific，#31430；Goat anti-Rabbit IgG(H+L)二抗购自Thermo Fisher Scientific，#31460)以1：20000稀释比室温孵育1h，PBST洗涤3遍每次10分钟。ECL显色。
如图4所示，免疫杂交(western blot)检测发现，UMI-77在HEK293T，Hela和U2OS细胞中以时间依赖的方式导致线粒体标记蛋白Tom20和Tim23降解，而不影响内质网标记蛋白Calnexin的降解。
2、实验方法：U2OS细胞以1.5*10 5个/ml的密度种于6孔板中，每孔2ml，5个孔。24小时后，分别在0小时，12小时，24小时，36小时加入UMI-77，终浓度为7.5μM。在48小时统一用250μl 2XSDS上样缓冲液收样。收样前3小时加入星形孢菌素终浓度1μM。100度加热10分钟。免疫杂交：每个样品上样10μl，电泳90V2h；转膜300mA，1h。5％脱脂牛奶封闭1h，抗体(Tom20抗体购自Cell Signaling Technology，#42406S；Caspase 3抗体购自Cell Signaling Technology，#9662；LC3B抗体购自Sigma-Aldrich，#L8918；LC3C抗体购自Proteintech，#18726-1-AP；Tubulin抗体购自杭州华安生物技术有限公司，#M1305-2)以一定稀释比(Tubulin抗体为1：5000，其余为1：1000)4度孵育过夜，PBST洗涤3遍每次10min。二抗(Goat anti-Mouse IgG(H+L)二抗购自Thermo Fisher Scientific，#31430；Goat anti-Rabbit IgG(H+L)二抗购自Thermo Fisher Scientific，#31460)以1：20000稀释比室温孵育1h，PBST洗涤3遍每次10分钟。ECL显色。
如图5所示，免疫杂交(western blot)检测发现，UMI-77在U2OS细胞中并没有诱导细胞凋亡关键蛋白Caspase3的激活。STS为星形孢菌素(Staurosporine)，是一种细胞凋亡诱导剂，在本实验中作为阳性对照。
3、实验方法：人胚肾转化细胞HEK293Tmtkeima细胞以1.5*10 5个/ml种于96孔黑色酶标板中，每孔100μl；24小时后加入UMI-77终浓度10μM，设置3个重复；于37度，5％CO 2培养条件下，处理6小时后，直接于biotek cytation3拍照。以469nm激发光做对焦通道，每孔拍摄4张图片，利用仪器软件处理图像。
如图6所示，验证人胚肾转化细胞HEK293Tmtkeima系统是否可以在UMI-77处理下诱导586nm激发荧光上升。UMI-77处理人胚肾转化细胞HEK293Tmtkeima细胞后，586nm激发的荧光明显上升，而NC(未作处理的)没有明显上升，因此，UMI-77可以诱导线粒体进入溶酶体发生线粒体 自噬。
4、实验方法：人胚肾转化细胞HEK293Tmtkeima细胞以1.5*10 5个/ml种于活细胞专用培养皿中，每皿2ml。24小时后用LysoGreen(溶酶体特异探针)(1000X)染色30分钟，替换新鲜培养基后，加入UMI-77，终浓度10μM，30分钟后置于Zesis LSM 880 with AiryScan激光共聚焦显微镜下活细胞培养，并连续拍摄1小时，每隔30秒拍一张照片。取240秒到450秒照片呈现。
如图7所示，加入UMI-77处理后线粒体被溶酶体包裹，发生共定位；没有处理的细胞没有任何共定位。
5、实验方法：HEK293T细胞以2*10 5个/ml的密度种于6孔板中，每孔2ml，5个孔。Hela细胞以1.5*10 5个/ml的密度种于6孔板中，每孔2ml，5个孔。24小时后，加入UMI-77，终浓度为5μM，处理24小时，在收样前12小时加入MG132 2μM，E64D 2μM和氯化铵20mM，Leupeptin(亮抑蛋白酶肽)50μM。在24小时统一用250μl 2XSDS上样缓冲液收样。100度加热10分钟。免疫杂交：每个样品上样10μl，电泳90V 2h；转膜300mA，1h。5％脱脂牛奶封闭1h，抗体(Tom20抗体购自Cell Signaling Technology，#42406S；Tim23抗体购自Proteintech，#11123-1-AP；Tubulin抗体购自杭州华安生物技术有限公司，#M1305-2)以一定稀释比(Tubulin抗体为1：5000，其余为1：1000)4度孵育过夜，PBST洗涤3遍每次10min。二抗(Goat anti-Mouse IgG(H+L)二抗购自Thermo Fisher Scientific，#31430；Goat anti-Rabbit IgG(H+L)二抗购自Thermo Fisher Scientific，#31460)以1：20000稀释比室温孵育1h，PBST洗涤3遍每次10分钟，ECL显色。
如图8所示，加入溶酶体抑制剂后阻止了UMI-77介导的线粒体标记蛋白降解，而蛋白酶体抑制剂MG132并没有阻止。
6、实验方法：HEK293T细胞以2*105个/ml的密度种于6孔板中，每孔2ml，5个孔。24小时后，加入UMI-77，终浓度为5μM，处理24小时，在收样前12小时加入E64D 2μM和氯化铵20mM，Leupeptin(亮抑蛋白酶肽)50μM。用戊二醛固定后进行电镜样品制备检测。
如图9所示，UMI-77处理后在溶酶体抑制剂存在情况下，明显观察到线粒体被自噬溶酶体包裹。
筛选得到的有效线粒自噬诱导剂UMI-77具有以下特点：
1)线粒体标记蛋白Tom20和Tim23降解，同时其他细胞器的标记蛋白不降解，如内质网的标记蛋白Calnexin不降解；
2)线粒体与溶酶体共定位，如用荧光标记线粒体和溶酶体，活细胞观察其共定位增加；
3)线粒体降解通过溶酶体通路，如加入溶酶体抑制剂E64D或氯化铵和Leupeptin(亮抑蛋白酶肽)可以阻止线粒体标记蛋白Tom20和Tim23的降解；
4)线粒体会被自噬溶酶体膜包裹，如用电镜观察可以看到多个线粒体被自噬溶酶体包裹。
实施例3线粒体自噬诱导剂UMI-77用于炎症的治疗的应用
1、实验方法：C57BL/6遗传背景的雄性4-8周龄小鼠购于南京模式动物中心。分为4组，每组6只，正常饲养1周适应后进行实验。全部药物溶于2％DMSO+30％PEG400+水的溶剂中。对每只小鼠称重，按照体重以UMI-77 30mg/kg，Nec1 2.5mg/kg腹腔注射药物200μl。LPS诱导组腹腔注射200μl溶剂。NC组不做处理。20分钟后注射LPS/D-Gal(脂多糖/D-半乳糖胺)浓度(LPS：100μg/kg，D-Gal：400mg/kg)。6小时后，眼球采血，并取出肝脏多聚甲醛固定。新鲜血液室温静置30分钟后，2000转每分钟，离心10分钟，取上清，用全自动生化分析仪测定ALT(谷丙转氨酶)，AST(谷草转氨酶)活力。肝脏固定后做HE(苏木精—伊红)染色。
如图10所示，LPS/D-Gal(脂多糖/D-半乳糖胺)诱导小鼠急性肝炎模型中，UMI-77可以显著抑制肝损伤。Nec1为已知的RIPK1蛋白抑制剂，UMI-77的效果好于Nec1。
如图11所示，LPS/D-Gal(脂多糖/D-半乳糖胺)诱导小鼠急性肝炎模型中，UMI-77可以显著抑制肝损伤。UMI-77恢复肝细胞的完整性。
实施例4 UMI-77通过诱导MCL1与LC3A(自噬标记物蛋白)相互作用诱导线粒体自噬
1、实验方法：
1)MCL1shRNA载体的构建。
根据网站https://dharmacon.horizondiscovery.com上设计两个MCL1  shRNA序列，并在两端加入BamHI和EcoRI酶切位点，送交上海生工生物合成。合成好的序列退火后连入pLV3载体形成pLV3-M2，pLV3-M5。
2)HEK293T细胞以2*10 5个/ml，Hela细胞以1.5*10 5个/ml的密度种于6孔板中，每孔2ml。每个细胞种6个孔。转染pLV3-M2，pLV3-M5入细胞，每个孔转入1μg shRNA质粒。转染方法：预先分别混合50μl无血清的DMEM培养基和1μg DNA，50μl无血清的DMEM培养基和2μl lip2000。最后将两个组分混合，室温静置20分钟，滴入培养基中。转染48小时后，加入UMI-77 5μM，处理12小时后统一用250μl 2X SDS上样缓冲液收样。100度加热10分钟。免疫杂交：每个样品上样10μl，电泳90V 2h；转膜300mA，1h。5％脱脂牛奶室温封闭1h，抗体(Tom20抗体购自Cell Signaling Technology，#42406S；Tim23抗体购自Proteintech，#11123-1-AP；Tubulin抗体购自杭州华安生物技术有限公司，#M1305-2，MCL1抗体购自杭州华安生物技术有限公司，#ET1606-14)以一定稀释比(Tubulin抗体为1：5000，其余为1：1000)4度孵育过夜，PBST(磷酸盐缓冲液+0.1％Tween 20)洗涤3遍每次10分钟。二抗(Goat anti-Mouse IgG(H+L)二抗购自Thermo Fisher Scientific，#31430；Goat anti-Rabbit IgG(H+L)二抗购自Thermo Fisher Scientific，#31460)以1：20000稀释比室温孵育1h，PBST洗涤3遍每次10分钟。ECL显色。
由图12可知，基因敲低实验后免疫杂交显示，MCL1蛋白被RNA干扰后表达量下降，此时再加入UMI-77后不能诱导线粒体标记蛋白Tom20和Tim23的降解。
2、实验方法：HEK293T细胞以2*10 5个/ml，接种于12孔板，每孔1ml，并预先在板中铺入免疫荧光载玻片。24小时后，加入UMI-77 10μM处理5小时，去除培养基，用PBS洗涤两遍，加入适量的4％多聚甲醛固定20分钟。用PBS洗涤两遍，用
Figure PCTCN2020083442-appb-000011
In Situ Red Starter Kit Mouse/Rabbit试剂盒(购自Sigma-Aldrich，#DUO92101)中的Blocking溶液封闭60分钟，37度。之后用上述试剂盒中的抗体稀释液稀释抗体(MCL1抗体购自santa cruz，#sc-69840，1：200稀释)，LC3A抗体购自Abcam，#ab62720，1：500稀释)，4度孵育过夜。用试剂盒中的洗涤试剂A洗涤片子2遍，每次10分钟。按照试剂盒的说明孵育二抗Anti-rabbit-PLUS和Anti-mouse-MINUS,37度60分钟。之后同样用洗涤试剂A洗涤片子2遍，按照试剂盒的说明用连 接酶37度连接30分钟。之后同样用洗涤试剂A洗涤片子2遍，按照试剂盒的说明等温扩增37度120分钟。最后用洗涤试剂B洗涤，用带有DNA染料DAPI的封片试剂封片。biotek Cytation3拍照，并用软件分析荧光强度和个数。
由图13可知，用
Figure PCTCN2020083442-appb-000012
In Situ Red Starter Kit Mouse/Rabbit试剂盒(购自Sigma-Aldrich，#DUO92101)在HEK293T细胞中检测内源的MCL1与LC3A相互作用。红色的点代表相互作用，红色点越多或者荧光强度变强说明其相互作用越强。UMI-77可以明显诱导红点变多，说明MCL1和LC3A相互作用增强。
3、实验方法：HEK293T细胞以2*10 5个/ml，接种于10cm培养皿。24小时后，共转染pCMV3-MCL1-3xFlag和pCDNA3.1-HA-LC3A或pCDNA3.1-HA-LC3B质粒，每个各4μg。转染方法：预先分别混合500μl无血清的DMEM培养基和8μg DNA，500μl无血清的DMEM培养基和16μl PEI(聚乙烯亚胺)转染试剂。最后将两个组分混合，室温静置20分钟，滴入培养基中。转染24小时后加入UMI-77 10μM处理5小时后，用PBS洗涤细胞两遍。用RIPA(20mM Tris-HCl,150mM NaCl,0.5％NP-40,1mM NaF,1mM Na 3VO 4,1mM EDTA)裂解液每个样品1ml，4度裂解30分钟。用枪头将细胞刮下，连同液体一同转移到1.5ml离心管中，4度12000g离心10分钟，取上清转移到新管中。从裂解好的上清中取出50μl样品加入等体积的2X SDS上样缓冲液收样，100度煮沸10分钟，作为Input样品。取适量的偶联有Flag抗体的琼脂糖凝胶，用RIPA裂解液洗涤3遍，每次2000rpm离心3分钟。将剩余的制备好的细胞裂解上清与预处理的偶联有Flag抗体的琼脂糖凝胶混合，4度混合过夜。第二天，2000rpm离心3分钟收取琼脂糖凝胶，并用RIPA洗涤3遍，每次2000rpm离心3分钟。最后收集琼脂糖凝胶用30μl的2X SDS上样缓冲液重悬，100度煮沸10分钟。免疫杂交：每个样品上样10μl，电泳90V 2h；转膜300mA，1h。5％脱脂牛奶室温封闭1h，抗体(Flag抗体购自杭州华安生物有限公司，#0912-1，HA抗体购自杭州华安生物有限公司，#0906-1)以1：5000稀释比4度孵育过夜，PBST(磷酸盐缓冲液+0.1％Tween 20)洗涤3遍每次10分钟。二抗(Goat anti-Rabbit IgG(H+L)二抗购自Thermo Fisher Scientific，#31460)以1：20000稀释比室温孵育1h，PBST洗涤3遍每次10分钟。ECL显色。
由图14可知，免疫共沉淀实验显示UMI-77诱导LC3A和MCL1相互作用增强。
4、实验方法：HEK293T细胞以2*10 5个/ml，接种于10cm培养皿。24小时后，共转染pCMV3-MCL1-3xFlag以及突变体和pCDNA3.1-HA-LC3A质粒，每个各4μg。转染方法：预先分别混合500μl无血清的DMEM培养基和8μg DNA，500μl无血清的DMEM培养基和16μl PEI(聚乙烯亚胺)转染试剂。最后将两个组分混合，室温静置20分钟，滴入培养基中。转染24小时后加入UMI-77 10μM处理5小时后，用PBS洗涤细胞两遍。用RIPA裂解液每个样品1ml，4度裂解30分钟。用枪头将细胞刮下，连同液体一同转移到1.5ml离心管中，4度12000g离心10分钟，取上清转移到新管中。从裂解好的上清中取出50μl样品加入等体积的2X SDS上样缓冲液收样，100度煮沸10分钟，作为Input样品。取适量的偶联有Flag抗体的琼脂糖凝胶，用RIPA裂解液洗涤3遍，每次2000rpm离心3分钟。将剩余的制备好的细胞裂解上清与预处理的偶联有Flag抗体的琼脂糖凝胶混合，4度混合过夜。第二天，2000rpm离心3分钟收取琼脂糖凝胶，并用RIPA洗涤3遍，每次2000rpm离心3分钟。最后收集琼脂糖凝胶用30μl的2X SDS上样缓冲液重悬，100度煮沸10分钟。免疫杂交：每个样品上样10μl，电泳90V 2h；转膜300mA，1h。5％脱脂牛奶室温封闭1h，抗体(Flag抗体购自杭州华安生物有限公司，#0912-1，HA抗体购自杭州华安生物有限公司，#0906-1)以1：5000稀释比4度孵育过夜，PBST(磷酸盐缓冲液+0.1％Tween 20)洗涤3遍每次10分钟。二抗(Goat anti-Rabbit IgG(H+L)二抗购自Thermo Fisher Scientific，#31460)以1：20000稀释比室温孵育1h，PBST洗涤3遍每次10分钟。ECL显色。
由图15可知，将MCL1含有的两个LIR motif分别突变。第一个LIR motif突变为四种形式：W261A,I264A,W261A/I264A,ΔLIR.第二个LIR motif突变为三种形式：F318A，V321A，F318A/V321A。第一个LIR motif突变后显著影响了MCL1和LC3A的相互作用。第二个LIR motif几乎没有影响。
实施例5 UMI77对小鼠炎症性肠病保护作用
实验方法：C57BL/6遗传背景的7-8周龄雄性野生型小鼠自由饮用2％DSS溶液(溶液需新鲜配制)6天后，改换饮用正常水至实验结束。实验 过程中，每天皮下注射UMI77(5mg/kg体重)；同时，每天记录小鼠体重变化、粪便形状和便血情况。实验中，粪便性状和便血情况评分参考文献Nat Protoc.2007；2(3):541-6；组织病理学评分分为炎性细胞浸润评分和组织损伤评分参考文献Immunity.2010；32(3):379-91，最终计算疾病活动指数。
实验结束，以CO 2处死小鼠后，离断自盲肠至肛门的全部肠段，拍照并测量长度。然后，取中间约1cm肠段放入包埋盒中浸入10％中性福尔马林以制备病理标本，用于HE染色；剩余肠段用50mL注射器以预冷的生理盐水进行冲洗，洗尽肠腔内剩余液体后，用小剪刀剪碎、混匀，锡箔纸包裹后液氮急冻，全部操作控制在小鼠死后10分钟以内完成。用Trizol裂解组织，按照Trizol说明书提取RNA；得到的RNA用ABI公司的逆转录试剂盒进行逆转录反应；SYBR Green法检测逆转录产物中炎症因子Cxcl1、G-CSF、IL6、IL-1β、S100A8和TNF-α，以管家基因Actin为内参，用2 -ΔΔCt法来计算和分析。
由图16-图21可知，UMI77可以显著性减缓体重下降幅度，降低疾病活动指数，恢复肠组织及其细胞正常状态，降低炎症因子表达，提示其可以保护炎症性肠病。
实施例6 UMI-77恢复APP/PS1模型老鼠的空间记忆学习能力
实验方法：目前通用的其中一个阿尔兹海默疾病的小鼠模型为APP/PS1小鼠。遗传背景为C57BL/6的APP/PS1小鼠和对照鼠购自于南京模式动物中心。分为三组，对照组，APP/PS1模型组，APP/PS1模型给药组，每组5-7只。APP/PS1 4月龄时开始腹腔注射给药UMI-77 13mg/kg。隔天给药，持续4个月，之后进行水迷宫实验。实验主要分为两部分：定位巡航实验和空间搜索实验。定位巡航实验用于测量小鼠对水迷宫的学习和记忆能力获取。空间搜索实验测量小鼠对平台空间位置记忆的保持能力。
定位巡航实验：将含有二氧化钛水的水池分为4个象限(西北，东北，西南，东南)，平台置于东南象限水下1cm左右。将小鼠头朝池壁放入水中，放入位置按照西北，东北，东南，西南的顺序放入4次。记录每次小鼠找到水下平台的时间。如果时间超过60秒，则引导小鼠到平台上，让其停留20秒，并记录此次找到平台时间为60秒。每只小鼠每天训练4次，一共训练4天，记录找到平台的时间(逃避潜伏期)。
由图22可知，颜色越深代表逃避潜伏期越长，越浅代表逃避潜伏期越短。从图中可以看到，随着训练时间的增加，每组小鼠的逃避潜伏期都有所下降，说明小鼠获得了空间位置的信息。但在训练过程中，APP/PS1小鼠的逃避潜伏前明显长于对照小鼠和给药小鼠，说明APP/PS1小鼠空间学习能力受损，在用UMI-77治疗后，能力恢复且优于野生型。总体上，用UMI-77治疗的APP/PS1小鼠的空间学习和记忆能力明显好于APP/PS1小鼠。(P<0.05,差异显著)。
空间搜索实验：定位巡航实验结束24小时后，将平台撤除，开始60秒的空间搜索实验。将小鼠由原先平台象限的对侧放入水中，记录小鼠穿过原先平台位置的次数和运动轨迹。
由图23可知，APP/PS1小鼠穿过原平台的次数显著低于对照小鼠，说明其空间记忆保持能力受损。(P<0.05,差异显著)。用UMI-77治疗后的APP/PS1小鼠的穿过次数明显多于APP/PS1小鼠。(P<0.01,差异显著)说明UMI-77可以明显提高APP/PS1小鼠的空间记忆保持能力。由图24可知，图中圆圈为原平台位置。对照组和给药组都有不同次数的穿过平台，APP/PS1小鼠并没有穿过平台。
阿尔兹海默症是一种严重的神经退行性疾病，目前并无特效药治疗。在之前的研究中发现阿尔兹海默症患者大脑中存在大量的淀粉样蛋白堆积和损伤线粒体堆积。目前已知的临床药物主要是针对淀粉样蛋白的清除和降解。UMI-77可以诱导线粒体自噬，帮助清除损伤线粒体，预示其对于治疗阿尔兹海默症的潜力。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. UMI-77作为线粒体自噬诱导剂在制备治疗炎症及神经退行性疾病药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述炎症为急性肝炎或者炎症性肠病。
  3. 根据权利要求1所述的应用，其特征在于，所述神经退行性疾病为阿尔兹海默症。
  4. 一种人胚肾转化细胞HEK293Tmtkeima稳定细胞株，其特征在于，该细胞株于2019年3月7日保藏于中国典型培养物保藏中心，保藏号为：CCTCC NO:C201940。
  5. 一种MCL1蛋白抑制剂的用途，其特征在于，用于制备药物或组合物，所述药物或组合物用于选自下组的一种或多种用途：
    (1)诱导线粒体自噬；
    (2)预防或抑制炎症；
    (3)预防或治疗炎症相关疾病；和
    (4)预防或治疗神经退行性疾病。
  6. 如权利要求5所述的用途，其特征在于，所述炎症包括选自下组的组织中的炎症：脂肪组织、肝脏组织、肌肉组织、皮肤组织、胰腺组织、肺组织、血管、心脏、骨骼、大脑、脊髓、视网膜、胃肠道组织、生殖系统，或其组合。
  7. 如权利要求5所述的用途，其特征在于，所述神经退行性疾病包括选自下组的疾病：阿尔茨海默病(AD)、帕金森病(PD)、亨廷顿病(HD)、肌萎缩性侧索硬化(ALS)、不同类型脊髓小脑共济失调(SCA)、和Pick病。
  8. 如权利要求5所述的用途，其特征在于，所述炎症相关疾病包括选自下组的疾病：急性肝炎、溃疡性肠炎、和自身免疫疾病。
  9. 如权利要求5所述的用途，其特征在于，所述MCL1蛋白抑制剂为结构如式I所示的化合物或其药学可接受的盐：
    Figure PCTCN2020083442-appb-100001
    其中，
    R1为
    Figure PCTCN2020083442-appb-100002
    X为卤素；
    R2为
    Figure PCTCN2020083442-appb-100003
    R 3为H、卤素、或羟基。
  10. 如权利要求6所述的用途，其特征在于，所述MCL1蛋白抑制剂为化合物UMI-77、化合物UMI-59或其药学可接受的盐：
    Figure PCTCN2020083442-appb-100004
  11. 如权利要求5所述的用途，其特征在于，所述药物或组合物含有安全有效量的MCL1蛋白抑制剂，和药学上可接受的载体。
  12. 一种诱导线粒体自噬的方法，其特征在于，包括步骤：给需要的对 象施用MCL1蛋白抑制剂。
  13. 一种促进MCL1蛋白和LC3A蛋白相互作用的方法，其特征在于，包括步骤：在包含MCL1蛋白和LC3A蛋白的体系中，施加MCL1蛋白抑制剂。
  14. 一种抑制炎症或治疗炎症相关疾病的组合物，其特征在于，所述组合物包括MCL1蛋白抑制剂。
  15. 一种预防或治疗神经退行性疾病的方法，其特征在于，所述方法包括步骤：给需要的对象施用安全有效量的MCL1蛋白抑制剂。
PCT/CN2020/083442 2019-05-09 2020-04-07 Mcl1蛋白抑制剂在制备治疗炎症及神经退行性疾病药物中的应用 Ceased WO2020224364A1 (zh)

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EP4406943A4 (en) * 2021-09-22 2025-09-10 Hangzhou Phecdamed Co Ltd SMALL MOLECULE COMPOUNDS HAVING A NAPHTHYLAMINE STRUCTURE AND THEIR USE
EP4406937A4 (en) * 2021-09-22 2025-09-10 Hangzhou Phecdamed Co Ltd SMALL MOLECULE COMPOUND HAVING A NAPHTHOL ETHER STRUCTURE AND ITS USE
WO2024192873A1 (zh) * 2023-03-20 2024-09-26 杭州天玑济世生物科技有限公司 一类具有萘胺结构的小分子化合物的用途
WO2024193528A1 (zh) * 2023-03-20 2024-09-26 杭州天玑济世生物科技有限公司 萘环类小分子化合物的用途

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